ABSTRACT Chronic pain is a major concern in public health with financial costs projected to surmount $600 billon in the next year. Patients afflicted with chronic pain endure extreme emotional, physical, and social burdens, resulting in severely diminished quality of life. Unfortunately, drugs currently used for chronic pain management, such as NSAIDs, opioids, neuronal stabilizers, and antidepressants, do not typically provide sufficient relief to restore full quality of life, and in many instances these treatments themselves limit patients, such as opioid treatment preventing a patient from legally driving. Recent preclinical studies have identified neuronal adenylyl cyclase type 1 (AC1) as a novel target for treating chronic pain. AC1 is highly expressed in neuronal tissues associated with pain processing and neuronal plasticity, and studies using AC1 knockout mice provide direct evidence linking AC1 to chronic inflammatory pain conditions. Furthermore, AC1 inhibitors would lack the side effects associated with other agents (e.g. opioids) used to treat chronic inflammatory pain. The development of AC1 inhibitors represents a unique challenge, as demonstrated by a prior preclinical AC1 inhibitor, NB001. NB001 has significant shortcomings, including modest selectivity over other adenylyl cyclase isoforms, likely due to its adenine-like structure. Compounds of this type are called P-site inhibitors and act by binding to the active site of AC that is conserved among all isoforms. Additional concerns for adenine-containing molecules like NB001 include effects on other cellular processes such as DNA synthesis. We hypothesize that developing a small molecule inhibitor of AC1 will allow us to mimic the AC1 knockout phenotype and provide a new avenue for the treatment of chronic inflammatory pain. We designed our studies to target NOT the conserved P-site or forskolin-binding site, but rather a novel approach, targeting the unique protein-protein interaction of AC1 and calmodulin (CaM). AC1 and AC8 are both activated by CaM, however, the CaM binding domains are unique in structure and location providing an unprecedented opportunity to achieve AC1 selectivity. Thus, the goals of this proposal are to: 1) develop a novel AC1/CaM biochemical screening assay, 2) implement this novel assay in a high throughput screen to interrogate a library of 100,000 compounds for inhibitors of the AC1/CaM protein- protein interaction, and 3) validate and chemically optimize lead molecules using cellular assays focused on selectivity and potency to guide medicinal chemistry efforts. To date, we have completed initial studies to develop the novel screening assay, established a subset of the necessary assays, and cemented the collaboration between the University of Iowa and Purdue University for the successful completion of our aims. We anticipate the identification of selective AC1 inhibitors that ultimately be improved and applied in models of chronic inflammatory pain.